The objective of the proposed study is elucidation of the molecular mechanisms which are involved in regulation of early and late transcription of Simian Virus 40. The significance of this area of research is that it bears directly on both mechanisms of regulation of eukaryotic genetic expression and the mechanism of transformation by the SV40 early gene. Over the past several years, certain aspects of the control early transcription have been elucidated; however, regulation of late transcription is poorly understood. Objective of the present proposal include elucidation of the role of three tandem 21 base-pair repeats, the main constituents of the viral early promoter, in early promotion; establishment of the mechanism by which large T antigen mediates a shift in early transcription initiation sites late in infection; identification of the promoter(s) for late transcription; investigation of a possible direct role for T antigen and sequences to which it binds in the regulation of late transcription; elucidation of the means by which interferon suppresses accumulation of early mRNAs; elucidation of the mechanism which leads to selective suppression of T antigen expression in teratocarcinoma stem cells which contain one copy of intact SV40 DNA and synthesize spliced early RNA; and establishment of a system for in vitro splicing of SV 40 and other eukaryotic RNAs. The first four objectives will be approahced, at least in part, by examining transcription either in vivo or in vitro of viral mutants with either deletions or base-substitutions in the 21 base pair repeats, T antigen binding sites and the region adjacent to the main late transcription initiation site. Studies concerned with the regulatory role of T antigen will in addition concentrate heavily on in vitro transcription of wild type and binding site mutant DNAs in the presence of increasing quantities of added T antigen and analysis of transcription in initiation sites. Emphasis in studies concerned with interferon action will be on distinguishing between decreased synthesis and increased degradation of early RNAs and seeing if accumulation of large and small T mRNAs are affected differentially. Analyses of mRNA 5' termini and splicing will be carried out by primer extension, a powerful method for determining the nucleo tide sequences of individual components of complex mixtures of RNAs.